Regiospecificity and stereospecificity of human udpglucuronosyltransferases in the glucuronidation of estriol, 16-epiestriol, 17-epiestriol, and 13-epiestradiols

Article abstract of DOI:10.1124/dmd.112.049072

The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B was examined. UGT1A10 is highly active in the conjugation of the 3-OH in all these estriols, whereas UGT2B7 is the most active UGT toward one of the ring D hydroxyls, the 16-OH in estriol and 16-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses indicated that the 17-OH configuration plays a major role in the affinity of UGT2B7 for estrogens. The glucuronidation of the different estriols by the human liver and intestine microsomes reflects the activity of UGT1A10 and UGT2B7 in combination with the tissues' difference in UGT1A10 expression. The UGT1A10 mutant 1A10-F93G exhibited much higher Vmax values than UGT1A10 in estriol and 17-epiestriol glucuronidation, but a significantly lower value in 16-epiestriol glucuronidation. To this study on estriol glucuronidation we have added experiments with 13-epiestradiol, a synthetic estradiol in which the spatial arrangement of the methyl on C18 and the hydroxyl on C17 is significantly different than in other estrogens. In comparison with estradiol glucuronidation, the C13 configuration change decreases the turnover of UGTs that conjugate the 3-OH, but increases it in UGTs that primarily conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol. The combined results reveal the strong preference of UGT1A10 for the 3-OH of physiologic estrogens and the equivalently strong preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of such steroid hormones. Copyright

Full text of DOI:10.1124/dmd.112.049072

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/01/03/dmd.112.049072.DC1.html
1
D
521-009X/41/3/582–591$25.00
RUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.112.049072
Drug Metab Dispos 41:582–591, March 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Regiospecificity and Stereospecificity of Human UDP-
Glucuronosyltransferases in the Glucuronidation of Estriol,
s
16-Epiestriol, 17-Epiestriol, and 13-Epiestradiol
Nina Sneitz, Mikko Vahermo, Johanna Mosorin, Liisa Laakkonen, Donald Poirier, and Moshe Finel
Centre for Drug Research (N.S., J.M., L.L., M.F.) and Division of Pharmaceutical Chemistry (N.S., M.V.), Faculty of Pharmacy,
University of Helsinki, Finland; and CHUQ-CHUL Research Center and Laval University, Quebec City, Québec, Canada (D.P.)
Received September 15, 2012; accepted January 3, 2013
ABSTRACT
The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the
value in 16-epiestriol glucuronidation. To this study on estriol
human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, glucuronidation we have added experiments with 13-epiestradiol,
and 2B was examined. UGT1A10 is highly active in the conjugation of a synthetic estradiol in which the spatial arrangement of the methyl
the 3-OH in all these estriols, whereas UGT2B7 is the most active on C18 and the hydroxyl on C17 is significantly different than in
UGT toward one of the ring D hydroxyls, the 16-OH in estriol and other estrogens. In comparison with estradiol glucuronidation, the
1
6-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses C13 configuration change decreases the turnover of UGTs that
indicated that the 17-OH configuration plays a major role in the conjugate the 3-OH, but increases it in UGTs that primarily
affinity of UGT2B7 for estrogens. The glucuronidation of the
different estriols by the human liver and intestine microsomes
reflects the activity of UGT1A10 and UGT2B7 in combination with
conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar
conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol.
The combined results reveal the strong preference of UGT1A10 for
the tissues’ difference in UGT1A10 expression. The UGT1A10 the 3-OH of physiologic estrogens and the equivalently strong
mutant 1A10-F93G exhibited much higher Vmax values than UGT1A10 preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of
in estriol and 17-epiestriol glucuronidation, but a significantly lower
such steroid hormones.
Introduction
estradiol glucuronidation, replacing phenylalanine 93 (F93) with
glycine (generating mutant 1A10-F93G) stimulates the estrogen
glucuronidation rate of UGT1A10 (Hoglund et al., 2011). These
findings prompted us to further investigate estrogen glucuronidation
by the human UGTs.
Estriol (16a,17b-estriol) is one of the three major endogenous estrogens,
along with estradiol and estrone (Fig. 1). There are two naturally occurring
stereoisomers of estriol, 16a,17a-estriol (17-epiestriol) and 16b,17b-
estriol (16-epiestriol) (Eliassen et al., 2009) (Fig. 1). Endogenously,
estriol is mainly generated from estradiol and estrone, but it can also
arise from 16-hydroxyandrostenedione, or from 16-hydroxydehydroe-
piandrosterone. The stereoisomer 17-epiestriol is formed by the
The UDP-glucuronosyltransferases (UGTs) are membrane enzymes
of the endoplasmic reticulum that catalyze the conjugation of different
aglycone substrates that carry suitable nucleophilic group or groups,
such as hydroxyl or amine, with glucuronic acid from the cosubstrate
UDP-glucuronic acid (Radominska-Pandya et al., 1999; King et al.,
000; Tukey and Strassburg, 2000).
Glucuronidation of endogenous estrogens was probably first
reported already in 1936 (Cohen and Marrian, 1936), and since then
the glucuronidation of estrogens has been extensively studied, both
in vitro and in vivo. We have examined the regioselectivity and
stereoselectivity of the 19 human UGTs of subfamilies 1A, 2A, and
2
2
B in the glucuronidation of 17b-estradiol (the natural estradiol), 17a- reduction of 16a-hydroxyestrone and 16-epiestriol in the oxidation
estradiol (17-epiestradiol), and enantiomeric b-estradiol (Itäaho et al., and reduction of estriol first to 16-ketoestradiol and subsequently 16-
008; Sneitz et al., 2011). One outcome of these studies was that as epiestriol (Longcope, 1984; Lappano et al., 2010) (Fig. 1).
long as the steroids contain a phenolic A ring (Fig. 1), UGTs 1A1,
Estriol has lower estrogenic potency than estradiol or estrone, and its
A3, 1A7, 1A8, and 1A10, mainly or exclusively, catalyze the circulating levels are relatively low in comparison with estradiol and
2
1
glucuronidation of the 3-OH of the estrogens (at variable rates), estrone (Ruggiero and Likis, 2002). During pregnancy, however, estriol
whereas the 17-OH of these steroid isomers is mainly conjugated by levels can rise considerably due to its production in the placenta from
one or more UGTs of subfamily 2B. In addition, we have recently 16-hydroxyandrostenedione and 16-hydroxydehydroepiandrosterone
shown that in the case of UGT1A10, the most active human enzyme in (Ruggiero and Likis, 2002; Lappano et al., 2010). Estriol can bind to
and activate estrogen receptors, but it also exerts antiestrogenic effects,
probably due to competitive binding that lowers the binding of the more
Financial support was obtained from the Sigrid Juselius Foundation; the
potent activator, estradiol to estrogen receptors (Melamed et al., 1997).
The physiologic properties of the estriol diastereomers 16- and 17-
epiestriol are poorly known. It has been reported that 17-epiestriol
Magnus Ehrnrooth Foundation; and the Academy of Finland [Grant 260010].
dx.doi.org/10.1124/dmd.112.049072.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: DMSO, dimethylsulfoxide; HIM, human intestine microsomes; HLM, human liver microsomes; HPLC, high-performance liquid
chromatography; UDPGA, uridine 59-diphosphoglucuronic acid-glucuronic acid; UGT, UDP-glucuronosyltransferase.
582

Glucuronidation of Estrogen Stereoisomers
583
uridine 59-diphosphoglucuronic acid (UDPGA) triammonium salt, UDPGA
trisodium salt, and alamethicin were purchased from Sigma-Aldrich (St. Louis,
MO). We obtained 16-epiestriol (3,16b,17b) from Steraloids (Newport, RI).
Deuterated methanol (d
epiestradiol (18-epiestradiol) was recently described elsewhere (Ayan et al.,
011).
Recombinant UGTs were expressed, as C-terminally-His-tagged proteins, in
4
) was from Sigma-Aldrich. The synthesis of 13-
2
baculovirus-infected Sf9 insect cells, and their relative expression levels were
determined as previously described elsewhere (Kurkela et al., 2003; Kurkela
et al., 2007). The F93G mutant of UGT1A10 was prepared as previously
described elsewhere (Hoglund et al., 2011). Most enzyme activity results in this
study, including the Vmax values, are presented as normalized values, meaning
that the measured rates were corrected according to the relative expression
levels of each UGT. Among the UGTs batches that were used in our study,
UGT1A3 had the lowest UGT expression level per mg of total protein in the
sample, and it was selected as the reference value, meaning a relative
expression level of 1.0. The relative expression levels of other UGT enzymes
were: UGT1A1, 1.6; UGT1A4, 3.7; UGT1A7, 15.5; UGT1A8, 1.4; UGT1A9,
1
.9; UGT1A10, 2.8; UGT1A10F93G, 2.3; UGT2A1, 2.4; UGT2A2, 6.7;
UGT2B4, 1.6; UGT2B7, 2.2 and 4.8; and UGT2B17, 1.7 and 2.4 (two different
batches of UGT2B7 and UGT2B17 were used). The only UGT for which
normalized rates could not be determined is UGT2B15, because in this case an
enzyme from a commercial supplier (BD Biosciences, Woburn, MA) that lacks
a C-terminal His-tag was used. Human liver microsomes (pool of 30 donors)
and human intestine microsomes (pool of 6 donors) were also purchased from
BD Biosciences.
Glucuronidation Assays with the Different Estrogens and Glucuronides
Chromatography. The glucuronidation assays were done in triplicate;
negative controls, namely, assays without UDPGA, were included in each set
of experiments. The substrate consumption during the different assays did not
exceed 10%. The results from the enzyme kinetics assays were analyzed with
GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA).
Estriol, 16- and 17-epiestriol, estradiol, and 13-epiestradiol incubation
mixtures contained 5 or 50 mg of UGT-enriched insect cell membranes or 0.5
or 10 mg of microsomes from either human liver (HLM) or intestine (HIM), 50
Fig. 1. Estriols structure and biosynthesis routes. 16-OH AD, 16-hydroxyandros-
tenedione; 16-OH DHEA, 16-hydroxydehydroepiandrosterone.
suppresses the expression of tumor necrosis factor a-induced vascular
cell adhesion molecule 1 (Mukherjee et al., 2003). In addition, the
mM phosphate buffer pH 7.4, 10 mM MgCl
2
, and 5 mM UDPGA
(
triammonium salt). Reaction mixtures with HLM and HIM also contained
urinary concentration of 17-epiestriol was found to positively correlate alamethicin, 5% of the total protein concentration. In the screening experi-
with the risk of breast cancer (Eliassen et al., 2012).
ments, performed at a single substrate concentration, the substrate concentra-
The human UGTs 1A1, 1A7, 1A9, 1A10, and 2B7 were previously tion was 200 mM in each case. In the enzyme kinetic assays, the aglycone
substrate concentration ranged from 0.2 to 300 mM. The substrates were
dissolved in dimethylsulfoxide (DMSO). The final DMSO concentration
in the reaction was 10%, and the total reaction volume was 100 ml. The
incubation times ranged from 15 to 60 minutes at 37°C, and the reactions
were terminated by the addition of 10 ml 4 M perchloric acid, transfer to ice,
and subsequent protein sedimentation by centrifugation (16,000g, 5 minutes).
The supernatants were analyzed by high-performance liquid chromatography
shown to catalyze estriol glucuronidation (Gall et al., 1999; Starlard-
Davenport et al., 2007). A comprehensive study on estriol glucuronidation,
one that identifies the conjugation position in estriol, or the type(s) of
estriol glucuronide(s) that is/are generated by each of the human
UGTs, has yet to be performed, however; even less is known about the
glucuronidation of 16-epiestriol and 17-epiestriol. The latter estrogen,
1
7-epiestriol, was reported to be a substrate for UGT2B4 and
(
HPLC), using a Shimadzu LC-10 model (Shimadzu Corporation, Kyoto,
UGT2B7, but that study did not include other human UGTs, making it Japan).
difficult to draw any conclusion about the UGT specificity of this
The three estriol glucuronides were separated using a Zorbax Eclipse Plus
steroid (Barre et al., 2007). No reports on 16-epiestriol glucuronidation 4.6 Â 150 mm, 5 mm column (Agilent Technologies, Palo Alto, CA) at
4
0°C. The eluents were 50 mM phosphate buffer pH 3.0 (A) and 1:1 (v/v)
have been published thus far.
mixture of acetonitrile/methanol (B). The following gradient was used: 0–37
minutes, 21% B; 37–38 minutes, B concentration increase to 50% B; 38–45
minutes 50% B; 45–50 minutes, B concentration decrease to 21%, at a flow
rate of 1.4 ml/min throughout. The detection of glucuronides and
unconjugated estriols was performed by use of a fluorescence detector at
A new estradiol diastereomer, 13-epiestradiol (previously called 18-
epiestradiol), has been synthesized, and its estrogenic activity has been
evaluated (Ayan et al., 2011). It was added to the current study
because it allows, for the first time, testing of how the configuration of
C13 and the spatial location of the methyl group 18CH3 affect
estrogen glucuronidation by individual UGTs.
2
1
80 nm (excitation) and 305 nm (emission). The retention times for the 3,
7, and 16 monoglucuronides of estriol were 7.4, 32.6, and 34.1 minutes,
The present study obtains significant new knowledge on the
respectively. They were identified and quantified by using authentic
glucuronidation of estrogens and further insights into the substrate glucuronide standards.
specificity of the human UGTs, mainly their regio- and stereo-
chemical preferences in estrogen glucuronidation.
The glucuronides of 17-epiestriol were also separated with Zorbax Eclipse
Plus 4.6 Â 150 mm 5 mm column at 40°C but using an isocratic
chromatography with mobile phase of 55% 50 mM phosphate buffer pH 3.0
and 45% methanol, at a flow rate of 1.3 ml/min flow. The detection was done
as in the case of estriol, and the retention times for the 3, 16, and 17
Materials and Methods
Estriol (3,16a,17b), 17-epiestriol (3,16a,17a), 17b-estradiol, estriol 3-b-D- monoglucuronides were 3.9, 6.8, and 11.5 minutes, respectively. The identity
glucuronide, estriol 16a-b-D-glucuronide, estriol 17b-b-D-glucuronide, of the 17-epiestriol 17-glucuronide was determined by NMR spectroscopy, and

5
84
Sneitz et al.
the glucuronides were quantified by use of an external standard curve generated estriol stereoisomers. For the 16-glucuronide and 17-glucuronide
with estriol glucuronides.
identification, however, we had no grounds to make corresponding
The glucuronides of 16-epiestriol were separated using a Poroshell 120 EC-
assumptions. To resolve this question and determine the identity of the
C18 4.6 Â 100 mm, 2.7 mM column (Agilent Technologies) at 40°C. The
glucuronide in one of the later-eluting glucuronide peaks, we turned to
isocratic mobile phase was composed of 55% 50 mM phosphate buffer pH 3.0
NMR spectroscopy. For the production of either the 16-glucuronide or
and 45% of 1:1 acetonitrile/methanol, at a flow rate of 0.8 ml/min. The
the 17-glucuronide of each of the two estriol stereoisomers, we have
employed UGT2B7. This enzyme was selected because preliminary
experiments revealed that incubation of UGT2B7 with either 16-
detection was performed the same as with estriol, and the retention times for the
3
, 17, and 16 monoglucuronides of 16-epiestriol were 3.7, 5.5, and 7.1 minutes,
respectively. The identity of 16-epiestriol 16-glucuronide was determined by
NMR, and the glucuronides were quantified by use of an external standard epiestriol or 17-epiestriol yields a large amount of a single, or nearly
curve generated with estriol glucuronides.
a single, glucuronide in each case (G3 in Fig. 2, B and C). These
Estradiol glucuronides were separated and quantified as previously described glucuronides were collected, purified, and subjected to structure
elsewhere (Itäaho et al., 2008). The glucuronides of 13-epiestradiol were
determination by NMR (see Materials and Methods for technical
details).
separated using a Chromolith SpeedRod RP18e column (50 Â 4.6 mm)
(
Merck, Darmstadt, Germany) at 40°C. The mobile phase consisted of 45%
Heteronuclear multiple-bond correlation NMR spectrum of the
purified glucuronide that UGT2B7 generates from 17-epiestriol
exhibited a clear correlation between the proton at C17 and the
anomeric carbon of the glucuronic acid moiety, as well as correlation
between the anomeric proton and C17 of 17-epiestriol. These results
50 mM phosphate buffer pH 3.0 and 55% MeOH, and the flow rate was 1.5
ml/min. Fluorescence detection was used (excitation 216 nm, emission 316
nm), and the retention times of 13-epiestradiol glucuronides were 2.2 and
6.1 minutes for the 3-glucuronide and 17-glucuronide of 13-epiestradiol,
respectively.
Biosynthesis and NMR Spectroscopy of Selected Estriol Glucuronides. indicate that when the substrate was 17-epiestriol, the UGT2B7-
The reaction mixtures for the biosyntheses contained 50 mM phosphate buffer produced glucuronide (G3 in Fig. 2C) is 17-epiestriol-17-glucuronide.
pH 7.4, 10 mM MgCl
2
, either 4 mg of 17-epiestriol or 8 mg of 16-epiestriol,
In the case of 16-epiestriol, NMR analysis of the main glucuronide
produced by UGT2B7 detected only a single correlation, between the
anomeric proton and C16 of 16-epiestriol (Table 1). Based on this, the
main glucuronide that UGT2B7 generates from 16-epiestriol was
identified as 16-epiestriol-16-glucuronide (G3 in Fig. 2B). Having
obtained these results, we concluded that the third glucuronide peak, the
one produced by UGT2B17 alongside 16-epiestriol-16-glucuronide
50 mM UDPGA (trisodium salt), 10% DMSO, and 7 ml of freshly prepared
homogenates of insect cells containing approximately 500 mg (total protein) of
UGT2B7-enriched insect cells in 25 ml. The biosynthesis reactions were
performed for 5 hours under continuous stirring at 37°C. The reactions were
terminated by the addition of 2.5 ml of ice-cold 4 M perchloric acid and transfer
to ice. Proteins were subsequently sedimented from the reaction mixture by
centrifugation (16,000g, 10 minutes), and the supernatants were fractionated
using an Agilent 1100 series HPLC (Agilent Technologies). The chroma-
(Fig. 3B), is 16-epiestriol-17-glucuronide. Similarly, it was concluded
tography methods for fractioning the resulting 16-epiestriol or 17-epiestriol that the small peak seen in 17-epiestriol glucuronidation by UGT2B7,
glucuronides were as described earlier for the screening analyses, except that the one that is the main peak in the case of 17-epiestriol glucuronidation
phosphate buffer was replaced with 0.1% HCOOH in water and the detection by UGT2A1 (Fig. 3C) is 17-epiestriol-17-glucuronide.
was by ultraviolet-detector at 280 nm. The glucuronide fractions were finally
Estriol Glucuronidation. Estriol glucuronidation by the different
concentrated using a rotary evaporator and lyophilized. The lyophilized
human UGTs, as well as the glucuronidation of 16-epiestriol and 17-
glucuronides were later redissolved in deuterated methanol and subjected to
epiestriol, was first investigated using a screening-type analysis in
NMR spectroscopy on a Varian 300 MHz MercuryPlus spectrometer (Varian,
which all the different UGTs were incubated with a single and rather
Inc., Palo Alto, CA). The chemical shifts of protons 16 and 17 of 16-
1
high-substrate concentration, 200 mM. The control experiments were
performed in the absence of either the aglycone substrate, estriol, or
the cosubstrate UDPGA. The determined glucuronidation rates for
epiestriol and 17-epiestriol glucuronides were assigned from H-NMR
spectra and the respective carbons from heteronuclear single quantum
coherence spectra. The chemical shifts of the anomeric proton and carbon of
the glucuronic acid moiety were similarly assigned. The site of glucur- each of the UGTs, with the exception of UGT2B15 (see Materials and
onidation was determined using heteronuclear multiple-bond correlation Methods), were corrected (normalized) according to their relative
spectra.
expression levels (see Materials and Methods).
The results of the estriol glucuronidation screening assays are
presented in Fig. 3A. Numerical values (Supplemental Table 1)
revealed that UGT1A10 and UGT2B7 are the most active enzymes
Results
Identification of the Glucuronides of the Different Estriols and toward this substrate, catalyzing its glucuronidation (at different
Estradiols. Estriols have three hydroxyl groups, and each of them hydroxyls) at as high a rate as 840 and 1200 pmol/min/mg,
may be conjugated but by different UGTs and at different rates. respectively (under the screening assays conditions). UGT1A10
Identifying the different glucuronides that are generated by the tested catalyzed estriol glucuronidation at the 3-OH, whereas UGT2B7
human UGTs from estriol (17b,16a-estriol) is relatively simple conjugated it exclusively at the 16-OH. UGTs 1A1 and 1A8 also
because commercial glucuronide samples for each of them are catalyzed estriol glucuronidation at the 3-OH, but only at low rates.
available. On the other hand, achieving a good separation between the UGTs 1A4, 2A1, and 2B4 catalyzed estriol conjugation at the 16-OH,
estriol-16- and 17-glucuronide peaks (G3 and G2, respectively, in but at low rates: 4–16 pmol/min/mg. UGT2B17 was the only human
Fig. 2A) by HPLC was challenging and required a long chromatog- UGT that catalyzed estriol glucuronidation at the 17-hydroxyl. The
raphy method.
glucuronidation rate of estriol by UGT2B17 was low, about 4 pmol/
In contrast to the situation with estriol, no authentic standards are min/mg, but its unique regioselectivity and/or stereoselectivity are of
currently available for any of the 16-epiestriol and 17-epiestriol interest.
glucuronides. Based on our results with estriol (Fig. 2) and the
Enzyme kinetic assays were performed with the two most active
previous results with estradiol and 17-epiestradiol (also called 17a- UGTs in estriol glucuronidation, 1A10 and 2B7, and the kinetic
estradiol or just epiestradiol in Itäaho et al., 2008), it is highly likely curves for both enzymes fitted best to the Michaelis-Menten equation
that the first eluting glucuronide of both 16-epiestriol and 17-epiestriol (Fig. 4). UGT2B7 exhibited only a slightly higher Vmax value than
(
G1 in Fig. 2, B and C), the main glucuronide products of UGT1A10 UGT1A10, apparently due to the high K
m
value of UGT1A10
in both cases (Fig. 2), are the 3-glucuronide of each of these (Table 2).

Glucuronidation of Estrogen Stereoisomers
585
Fig. 2. Chromatographic separation of the different glucuronides of the three estriol stereoisomers. The glucuronide peaks are (A) authentic standards (estriol) or (B)
generated from incubation with UGT1A10, 2B7, and 2B17 (16-epiestriol) or (C) UGT1A10 and 2B7 (17-epiestriol). A negative control, from the incubation of estriol with
UGT1A10 in the absence of UGPGA, is presented at the bottom of panel A. The detected glucuronides, G1–G3, are numbered according to their elution order and are color-
coded according to the glucuronidation site. Black represents 3-glucuronides, blue 17-glucuronides, and red 16-glucuronides. The numeric results of the screening assays are
presented in Supplemental Table 1.
1
6-Epiestriol Glucuronidation. The initial screening experiment for glucuronides identification). The 16-epiestriol glucuronidation rates of
6-epiestriol glucuronidation demonstrated that, as with estriol glucur- UGT1A10 and UGT2B7 were 3500 and 2700 pmol/min/mg, re-
onidation, UGTs 1A10 (3-OH conjugation, see Discussion) and 2B7 spectively. UGTs 1A1, 1A7, 1A8, and 2B15 catalyzed the formation of
16-OH conjugation) are the most active enzymes (see earlier for the 16-epiestriol-3-glucuronide but at much lower rates, only 0.5–16.0
1
(

5
86
Sneitz et al.
TABLE 1
catalyzed a relatively high rate of 16-epiestriol glucuronidation,
approximately 260 pmol/min/mg, whereas the activities of UGTs 1A3,
Nuclear magnetic resonance analyses: 1H and 13C chemical shifts
2
B4, and 2B17 were significantly lower: 5-45 pmol/min/mg.
The enzyme kinetics of 16-epiestriol glucuronidation curves by
16-Epiestriol Glucuronide
17-Epiestriol Glucuronide
H16
H17
C16
C17
H (anomeric)
C (anomeric)
4.18 (m)
3.79 (d,J = 9.7 Hz)
80.5
4.48 (m)
3.79 (d,J = 5.1 Hz)
73.3
UGT1A10 and UGT2B7 fitted best to the Michaelis-Menten equation
with substrate inhibition (Fig. 4; Table 2). The Vmax and K values for
both these UGTs suggested low affinity for 16-epiestradiol but high
turnover rates.
m
76.5
88.9
4.46 (d,J = 7.9 Hz)
106.0
4.42 (d,J = 7.5 Hz)
104.7
1
7-Epiestriol Glucuronidation. UGT2B7 exhibited the highest
rate of 17-epiestriol glucuronidation, preferentially (but not exclu-
sively) conjugating the 17-OH, not the 16-OH as in estriol and 16-
pmol/min/mg. Besides UGT2B7, UGTs 1A3, 2A1, 2B4, and 2B17 epiestriol (Fig. 3). The other enzyme that catalyzes the conjugation of
catalyzed the formation of 16-epiestriol-16-glucuronide. The 16- the 17-OH of 17-epiestriol is UGT2B4, an enzyme that often exhibits
epiestriol-16-glucuronide formation by UGT1A4 was barely detectable, similar substrate selectivity to that of UGT2B7 but with lower turnover
as was the 16-epiestriol-17-glucuronide formation by UGT2A1. UGT2A1 rates. UGT2A1 generated some 17-epiestradiol-17-glucuronide but at
Fig. 3. Glucuronidation rates of the different estriol
diastereomers (see Fig. 1 for structures) by the
human UGTs at a single substrate concentration,
2
00 mM. The glucuronidation rate values for estriol
(A), 16-epiestriol (B), and 17-epiestriol (C) are
expressed as the mean of triplicate samples 6 S.E.
The glucuronidation rates of all the UGTs, with the
exception of UGT2B15 (indicated by *), were
corrected according to their relative expression
values (normalized, see Materials and Methods).

Glucuronidation of Estrogen Stereoisomers
587
Fig. 4. Enzyme kinetics of the glucuronidation of estri-
ol, 16-epiestriol, and 17-epiestriol by UGT1A10 and
UGT2B7. The analyses were performed in triplicate, and
the rates were normalized according to the relative ex-
pression levels of the UGTs. The derived kinetic
constants are presented in Table 2. The Eadie-Hofstee
transformations of the curves are shown in Supplemental
Fig. 1.
a far lower rate than it generated 17-epiestradiol-16-glucuronide, while
UGT2B7 also catalyzed 17-epiestradiol-16-glucuronide formation
UGT2A2 produced trace amounts of 17-epiestradiol-17-glucuronide but at a much lower rate than 17-epiestriol-17-glucuronide formation.
(
Fig. 3; Supplemental Table 1).
The only UGT that catalyzed rather high rates of 17-epiestriol-16-
glucuronide formation was the extrahepatic UGT2A1. UGT1A4
produced some 17-estriol-16-glucuronide but at a very low rate (Fig.
TABLE 2
Enzyme kinetic parameters for the glucuronidation of estriol, 16-epiestriol, and 17-
epiestriol by UGTs 1A10 and 2B7 as well as the mutant 1A10-F93G
3
; Supplemental Table 1).
UGT1A10 exhibited the highest rate of 17-epiestriol-3-glucuronide
Formation of the three glucuronides (g) from the different substrates was assayed in the cases
of UGT1A10 and the mutant 1A10-F93G, whereas formation of 16-glucuronide by UGT2B7 was
assayed when the substrate was estriol or 16-epiestriol; formation of 17-glucuronide by UGT2B7
was assayed when the substrate was 17-epiestriol.
formation (see Discussion), even if this rate was much lower than the
rate at which UGT1A10 catalyzes the conjugation of the 3-OH in
either estriol or 16-epiestriol (Fig. 3). The other UGTs that exhibit
detectable activity in the glucuronidation of 17-epiestriol at the 3-OH
were 1A1 (low), 1A3 (low), 1A7 (traces), 1A8, 2A1 (traces), and
2B15 (Fig. 3; Supplemental Table 1).
Enzyme kinetic assays of 17-epiestriol glucuronidation were
performed with UGT1A10 and UGT2B7. The kinetic curves fit-
ted best to the Michaelis-Menten equation in both cases (Fig. 4),
even if the low affinity of UGT1A10 for this substrate, in
combination with the limited water solubility of estrogens, makes
it difficult get a fully reliable analysis for this enzyme. The Vmax
values of both enzymes were rather similar while their Km
values differed fundamentally (Table 2). It is particularly worth
UGT1A10 (3-g) 1A10-F93G (3-g) UGT2B7 (16-g or 17-g)
Estriol
(16-g)
1509 6 27.7
10.3 6 0.8
(16-g)
3866 6 788
38.4 6 11.4
162 6 86.4
(17-g)
V
max (pmol/min/mg) 1200 6 28.1 7535 6 618
K
m
(mM)
68.4 6 4.7
230 6 29.7
1
6-epiestriol
max (pmol/min/mg) 7700 6 740
V
2249 6 201
186 6 28.0
K
K
m
(mM)
(mM)
59.8 6 8.0
98.1 6 16.6
a
i
1
7-epiestriol
max (pmol/min/mg)
(mM)
V
K
958 6 75.0 5712 6 216
337 6 42.1 51.1 6 5.4
820 6 18.3
0.6 6 0.1
m
UGTs, UDP-glucuronosyltransferases.
a
K
i
values are indicated when the kinetic curves fit best to the Michaelis-Menten with
substrate inhibition model.
m
noting the apparent K value of UGT2B7 for this substrate, only 0.6

5
88
Sneitz et al.
6
0.1 mM, which is rather uncommonly low among the human increased Vmax value of the mutant, not from a decreased Km value
UGTs.
(Table 2). The results with the two epiestriols, however, were
Estriol Diastereomers Glucuronidation by Human Liver and somewhat unexpected (Supplemental Fig. 3; Table 2). They revealed
Intestine Microsomes. Estriol stereoisomer glucuronidation by HLM that the glucuronidation activities of this mutant are strongly affected
and HIM was examined as well. The results (Fig. 5) are in good by the configuration of C16 and C17 of estriol, but in different ways
agreement with the results for individual UGTs when the tissue- than wild-type UGT1A10 was affected by them. In the case of 16-
specific expression pattern of the UGTs, particularly UGT2B7, epiestriol, the mutant exhibited much lower Vmax and higher K
UGT1A10, and UGT2B4, is taken into account, as will be discussed. than wild-type UGT1A10, whereas in the case of 17-epiestriol the
Effect of the F93G Mutation on the Glucuronidation of Estriol mutant’s activity was very high due to both lower K and higher Vmax
Diastereomers by UGT1A10. In a recent study we found that values those of the wild-type enzyme (Supplemental Fig. 3; Table 2).
replacing phenylalanine 93 with a glycine residue, the amino acid with 13-Epiestradiol Glucuronidation. Our previous and current results
m
values
m
no side chain, strongly stimulated the estriol and ethinylestradiol have suggested that the configuration of the D-ring carbons C16 and
glucuronidation activity of UGT1A10 (Höglund et al., 2011). We have C17 strongly affect the glucuronidation of estrogens by the different
now examined the activity and kinetics of the 1A10-F93G mutant in human UGTs. Nevertheless, a closer inspection of the results,
the glucuronidation of 16-epiestriol and 17-estriol, in comparison with particularly the conjugation site by UGT2B7, raises the possibility
the wild-type UGT1A10 and with the estriol glucuronidation activity. that the spatial relations between the 17-OH and the methyl group on
The strong stimulation of the estriol glucuronidation rate by the C13, a carbon that belongs to both ring C and ring D, also affects the
1
A10-F93G mutant (Supplemental Fig. 3) was nearly the same as in results. In particular, the only case where UGT2B7 exhibited high
the previous study (Höglund et al., 2011), and it stems mainly from the glucuronidation rates and high substrate affinity was 17-epiestriol, the
Fig. 5. Enzyme kinetics of estriol, 16-epiestriol, and
1
7-epiestriol glucuronidation by HLM and HIM. d, 3-
glucuronide; j, 16-glucuronide; ., 17-glucuronide.
The Eadie-Hofstee transformations of the curves are
shown in Supplemental Fig. 2.

Glucuronidation of Estrogen Stereoisomers
589
only substrate in this study in which C13 and C17, which are bonded in which the C17 is in the b configuration (Itäaho et al., 2008; Sneitz
to each other in ring D (Fig. 6), are in opposite configurations. Due to et al., 2011). In the case of 13-epiestradiol, however, UGT2B17
this, when the synthetic estradiol isomer 13-epiestradiol (Fig. 6) catalyzes the conjugation of the 3-OH at somewhat faster rate than the
became available (Ayan et al., 2011), we included it in this study and conjugation of the 17-OH (Fig. 7).
screened the UGTs for the glucuronidation of this estrogen. The
One more unexpected result with 13-epiestradiol was the formation
results with 13-epiestradiol were compared with the glucuronidation of some glucuronides, let alone both glucuronides, even by UGT1A9,
of estradiol (17b-estradiol) using the very same batches of the an enzyme that has not exhibited estrogen glucuronidation before even
different recombinant UGTs.
if we have demonstrated that it binds estradiol (Itäaho et al., 2008). We
The two different glucuronides of 17b-estradiol, the main physio- have also tested estradiol and 13-epiestradiol glucuronidation by the
logic estradiol isomer, were identified using authentic standards. No F93G mutant of UGT1A10. In agreement with previous results (Höglund
authentic glucuronide standards were available for 13-epiestradiol, et al., 2011) and in contrast to the estriol glucuronidation activity, the
however. As noted earlier, our previous results indicated that the first F93G mutation did not stimulate estradiol glucuronidation by
eluting glucuronide, the produced by UGT1A10 and UGT1A1, is the UGT1A10. On the other hand, the 13-epiestradiol glucuronidation
3
1
-glucuronide. It follows that the second eluting glucuronide of rate of the 1A10-F93G mutant was about 4-fold higher than the
3-epiestradiol, the main product of 13-epiestradiol glucuronidation corresponding activity of wild-type UGT1A10 (Fig. 7).
by UGT2A1 and the only product of UGT2B7 in this case, is 13-
epiestradiol-17-glucuronide.
The screening results suggest that the a-configuration of C13
significantly lowers the rates of the UGT1A10 catalyzed glucuroni-
Discussion
Estrogens and androgens regulate important functions and gene
dation reaction as well as the activity of the other UGTs of subfamily expression in the body (Katzenellenbogen, 1996). From the point of
A that mainly catalyze estradiol conjugation at the 3-OH, namely, view of UGTs research, steroids are also an interesting group of
1
UGTs 1A1, 1A3, and 1A8. (Fig. 7). On the other hand, the change in compounds for the study of substrate specificity due to their rigid
the configuration of C13 from b in the regular estradiol to a in 13- backbone structure and the availability of several regio-isomers and
epiestradiol appears to stimulate or even change the activities of many stereo-isomers. Estriol, 16-epiestriol, and 17-epiestriol are all built of
UGTs of subfamilies 2A and 2B. In addition, it also stimulates four rings, and the main difference between them is in ring D (Fig. 1).
somewhat the low activity of UGT1A4, the only member of the To extract information on the substrate specificity of the different
UGT1A subfamily that specifically conjugates estrogens at the 17- UGTs, one has to identify the site of glucuronidation in each case, an
OH, even if at low rates (Fig. 7).
important but sometimes challenging objective. In this study, we have
Both UGTs 2A1 and 2A2 catalyzed the formation of the two 13- partly overcome this challenge by purification of two glucuronides and
epiestradiol glucuronides at much higher respective rates than the identification of the conjugation site in them by use of NMR (Table 1).
formation of the corresponding glucuronides of 17b-estradiol (Fig. 7).
Our results demonstrate that UGT1A10 is the most active enzyme
The rates of both the 3- and 17-glucuronide formation from 13- in the conjugation of the 3-OH in each of these estriol diastereomers. It
epiestradiol by UGT2B15 were also much higher than the corre- may be noted that the assignment of the conjugation site of UGT1A10
sponding rates when the substrate was 17b-estradiol (Fig. 7). In the in estriol was based on the migration of the authentic estriol-3-
case of UGT2B7, only the formation of the 13-epiestradiol-17- glucuronide standard. No such standards are available for the two
glucuronide was observed, but the rate of this activity was increased in epiestriols, but there are good reasons to assume that the glucuronides
comparison with 17b-estradiol. Moreover, although no 17b-estradiol generated by UGT1A10 from either 16-epiestriol or 17-epiestriol, the
glucuronidation by UGT2B4 was observed, it catalyzed measurable glucuronide that is eluted first under our chromatography conditions,
rates of 13-epiestradiol glucuronidation at the 17-OH (Fig. 7). Perhaps is the corresponding 3-glucuronide This assignment is based on the
the most significant change between the glucuronidation of the two similarity of epiestriols in physicochemical properties to estriol (for
estradiols that were tested here was with UGT2B17, an enzyme that is which authentic standards are available) and the similarity of such
mostly specific for the 17-OH in 17b-estradiol and in all the estrogens properties among their 3-glucuronides (the differences among the 16-
and 17-glucuronides are likely to be much larger). The second reason
for the suggested assignment is the tendency of UGT1A10 to mainly
conjugate the phenolic hydroxyl in estrogens, the 3-OH (or other
phenolic hydroxyls in ring A, if present), rather than the 16-OH or the
1
7-OH that are secondary hydroxyls (Fig. 3A of this study for estriol;
Itäaho et al., 2008 for estradiol; Lepine et al., 2004 for estrone,
estradiol, and hydroxyestradiols). These reasons and the NMR results
for the ring D glucuronide that is generated by UGT2B7 (Table 1) give
strong support to the assignment of the glucuronidation sites within
the estrogens that were tested in this study.
UGT2B7 is clearly the most active enzyme in the conjugation of
one of the two hydroxyls on ring D of the estriols (Fig. 3). In addition
to the glucuronidation site, UGT1A10 and UGT2B7 also differ
considerably from each other in their affinity toward the different
estriols, particularly toward 17-epiestriol (Fig. 4). Nevertheless, both
enzymes exhibited very high glucuronidation rates toward 16-
Fig. 6. Structures of estradiol (A) and 13-epiestradiol (B) in both two-dimensional epiestriol while the lowest rate of both enzymes was toward 17-
(
upper panels) and space-filling modes (lower panels). The different rings of the
epiestriol (Fig. 3; Table 2).
Considering that the expression levels of UGT1A10 and UGT2A1
in HLM are very low (Ohno and Nakajin, 2009; Sneitz et al., 2009;
steroids’ backbone are indicated in the two-dimensional presentation (upper panels),
along with carbons 3, 13, 17, and 18 (both upper and lower panels). The oxygen
atoms are shown in red in the space-filling models.

5
90
Sneitz et al.
TABLE 3
Enzyme kinetic parameters for the glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by human liver microsomes
(HLM) and human intestine microsomes (HIM)
HLM
HIM
16-g
3
-g
16-g
17-g
3-g
17-g
Estriol
V
K
max (pmol/min/mg)
95 6 45
313 6 214
4347 6 50
13 6 0.7
—
—
839 6 19
75 6 4
953 6 21
5.1 6 0.7
2095 6 947
1119 6 575
m
(mM)
1
6-epiestriol
max (pmol/min/mg)
(mM)
7-epiestriol
max (pmol/min/mg)
(mM)
a
V
K
569 6 1144 8011 6 106
;8400a
;5300
4111 6 94
50 6 3
1480 6 35
43 6 3
2324 6 1107
1168 6 629
m
2375 6 5094
33 6 1
1
V
K
805 6 157
380 6 102
128 6 6
7.8 6 2
5425 6 67 1239 6 150
21 6 1 251 6 47
266 6 394
820 6 1438
1633 6 13
1.3 6 0.14
m
a
An estimate, as accurate kinetic parameters could not be determined.
Court et al., 2012), the results with HLM (Fig. 5) suggest that support this property of UGT2B7. The results with the other two
UGT2B7 plays a major role in the glucuronidation of the three estriols estriols suggest that when the estriol’s C17 is in the b configuration,
in the liver. The kinetic results (Fig. 5) also suggest that another UGT2B7 only conjugates the 16-OH, regardless of whether the C16 is
enzyme, perhaps UGT2B4 which is highly expressed in the liver but in the a (estriol) or the b (16-epiestriol) configuration (Fig. 3). This
not in the intestine (Ohno and Nakajin, 2009; Court et al., 2012), suggests that the configuration of C17 in either estriol or estradiol
contributes to 17-epiestriol glucuronidation on the 17-OH in the liver. plays a dominant role in determining the affinity of UGT2B7 for
One of the main reason to undertake a detailed study on estriols estrogens. The only case so far in which C17 of estradiol was in the a
m
glucuronidation by the human UGTs was a previous result with configuration and the UGT2B7 K for the substrate was not very low
estradiol and 17-epiestradiol according to which the affinity of was enantio-estradiol glucuronidation (Sneitz et al., 2011), a result
UGT2B7 for the estrogen substrate is strongly increased when the that may be due to the configuration of the other chiral centers in
configuration of C17 is a rather than b (Itäaho et al., 2008). The new the substrate molecule. Hence, it might be suggested that the a
results on the kinetics of 17-epiestriol by UGT2B7 (Fig. 4) fully configuration of C17 has such an effect on UGT2B7 because it
Fig. 7. Glucuronidation of estradiol (A) and 13-
epiestradiol (B) (see Fig. 6 for structures) by the
human UGTs. The screening assay was performed
at a single substrate concentration, 200 mM. The
rates, the mean of triplicate assays with S.E., were
normalized according to the relative expression
levels (see Materials and Methods) and are
presented on a scale. The numeric results are
presented in Supplemental Table 1. Note that the
Y-axis values are presented on a logarithmic
scale.

Glucuronidation of Estrogen Stereoisomers
591
changes the spatial relations between 17-OH and the methyl group on Authorship Contributions
C18. It was not easy to find a good substrate for testing the latter
suggestion, and the reason 13-epiestradiol was added to the current
study was because it was the only possible currently available estrogen
in which the spatial organization of the 17-OH and 18-CH3 is
considerably different than in other estrogens. Nevertheless, even 13-
epiestradiol does not provide a very good test compound because the
conformational change in C13 leads to larger changes in the structure
of the entire estrogen molecule (Fig. 6).
Participated in research design: Sneitz, Finel.
Conducted experiments: Sneitz, Mosorin, Vahermo.
Contributed new reagents or analytic tools: Poirier, Vahermo.
Performed data analysis: Sneitz, Laakkonen, Vahermo.
Wrote or contributed to the writing of the manuscript: Sneitz, Finel.
References
Ayan D, Roy J, Maltais R, and Poirier D (2011) Impact of estradiol structural modifications (18-
methyl and/or 17-hydroxy inversion of configuration) on the in vitro and in vivo estrogenic
activity. J Steroid Biochem Mol Biol 127:324–330.
Barre L, Fournel-Gigleux S, Finel M, Netter P, Magdalou J, and Ouzzine M (2007) Substrate
specificity of the human UDP-glucuronosyltransferase UGT2B4 and UGT2B7. Identification
of a critical aromatic amino acid residue at position 33. FEBS J 274:1256–1264.
Cohen SL and Marrian GF (1936) The isolation and identification of a combined form of oestriol
in human pregnancy urine. Biochem J 30:57–65.
While the a configuration of C17 strongly increases the affinity of
UGT2B7 for estrogens, in the case of UGT1A10 it lowers the
enzyme’s affinity for estrogens, as far as can be judged from the K
m
values (Table 2). The configuration of the estriols’ C17 and/or C16
also affects the activity of UGT2B15 in estrogen glucuronidation,
except that the rates exhibited by this enzyme are relatively low and
the effects are more complex. When C17 of estradiol (17a-estradiol,
Itäaho et al., 2008, as well as enantio-estradiol, Sneitz et al., 2011) or
estriol (17-epiestriol, our study) is in the a configuration, UGT2B15
conjugates the 3-OH, the only member of the UGTs subfamily
Court MH, Zhang X, Ding X, Yee KK, Hesse LM, and Finel M (2012) Quantitative distribution
of mRNAs encoding the 19 human UDP-glucuronosyltransferase enzymes in 26 adult and 3
fetal tissues. Xenobiotica 42:266–277.
Eliassen AH, Spiegelman D, Xu X, Keefer LK, Veenstra TD, Barbieri RL, Willett WC, Han-
kinson SE, and Ziegler RG (2012) Urinary estrogens and estrogen metabolites and subsequent
risk of breast cancer among premenopausal women. Cancer Res 72:696–706.
Eliassen AH, Ziegler RG, Rosner B, Veenstra TD, Roman JM, Xu X, and Hankinson SE (2009)
Reproducibility of fifteen urinary estrogens and estrogen metabolites over a 2- to 3-year period
in premenopausal women. Cancer Epidemiol Biomarkers Prev 18:2860–2868.
Gall WE, Zawada G, Mojarrabi B, Tephly TR, Green MD, Coffman BL, Mackenzie PI,
and Radominska-Pandya A (1999) Differential glucuronidation of bile acids, androgens and
estrogens by human UGT1A3 and 2B7. J Steroid Biochem Mol Biol 70:101–108.
Höglund C, Sneitz N, Radominska-Pandya A, Laakonen L, and Finel M (2011) Phenylalanine 93
of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens.
Steroids 76:1465–1473.
Itäaho K, Mackenzie PI, Ikushiro S, Miners JO, and Finel M (2008) The configuration of the 17-
hydroxy group variably influences the glucuronidation of beta-estradiol and epiestradiol by
human UDP-glucuronosyltransferases. Drug Metab Dispos 36:2307–2315.
Katzenellenbogen BS (1996) Estrogen receptors: bioactivities and interactions with cell signaling
pathways. Biol Reprod 54:287–293.
2
B to do so, or at least this was our conclusion until testing the
glucuronidation of 13-epiestradiol (Fig. 7). In the case of UGT1A3, the
configuration change in C16 of estriol from a to b stimulates
conjugation but at the 16-OH, not at the 3-OH as it does when the
substrate is 17-epiestriol (Fig. 3B), estradiol, or 13-epiestradiol (Fig. 7).
UGT2B17, like UGT2B15, exhibited low rates in the glucuronida-
tion of the different estriols but an interesting stereoselectivity. As with
estradiols (Itäaho et al., 2008), UGT2B17 only conjugates the 17-OH of
estrogens when it is located “above” the plane of ring D. UGT2B17
failed completely to glucuronidate 17-epiestriol, the variant in which
both C17 and C16 are in the a configuration (Fig. 1). In the case of 16-
epiestriol, where both the C16 and C17 are in the b configuration,
UGT2B17 conjugated both of them at similar rates (Fig. 3).
King CD, Rios GR, Green MD, and Tephly TR (2000) UDP-glucuronosyltransferases. Curr Drug
Metab 1:143–161.
Kurkela M, García-Horsman JA, Luukkanen L, Mörsky S, Taskinen J, Baumann M, Kostiainen
R, Hirvonen J, and Finel M (2003) Expression and characterization of recombinant human
UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than
other UGTs and was purified as an active dimeric enzyme. J Biol Chem 278:3536–3544.
Kurkela M, Patana AS, Mackenzie PI, Court MH, Tate CG, Hirvonen J, Goldman A, and Finel M
(
2007) Interactions with other human UDP-glucuronosyltransferases attenuate the con-
Aside from the glucuronidation site, the results also show that the
glucuronidation rate of the different estriols depends on the configu-
ration of C16 and/or C17, even if the conjugation target is the 3-OH, as
in UGT1A10 (Fig. 3). In this respect, it is interesting that a point
mutation in UGT1A10, F93G, largely changes the relation between
the structure of the estriol and its glucuronidation rate. The findings
with mutant 1A10-F93G also suggest that the differences in the
glucuronidation rate of the three estriols could not be assigned to
a small difference in the physicochemical properties among them.
The configuration change at C13 of estradiol changes the spatial
sequences of the Y485D mutation on the activity and substrate affinity of UGT1A6. Phar-
macogenet Genomics 17:115–126.
Lappano R, Rosano C, De Marco P, De Francesco EM, Pezzi V, and Maggiolini M (2010) Estriol
acts as a GPR30 antagonist in estrogen receptor-negative breast cancer cells. Mol Cell Endo-
crinol 320:162–170.
Lépine J, Bernard O, Plante M, Têtu B, Pelletier G, Labrie F, Bélanger A, and Guillemette C
(2004) Specificity and regioselectivity of the conjugation of estradiol, estrone, and their cat-
echolestrogen and methoxyestrogen metabolites by human uridine diphospho-
glucuronosyltransferases expressed in endometrium. J Clin Endocrinol Metab 89:5222–5232.
Longcope C (1984) Estriol production and metabolism in normal women. J Steroid Biochem 20
(
4B):959–962.
Melamed M, Castaño E, Notides AC, and Sasson S (1997) Molecular and kinetic basis for the
mixed agonist/antagonist activity of estriol. Mol Endocrinol 11:1868–1878.
Mukherjee TK, Nathan L, Dinh H, Reddy ST, and Chaudhuri G (2003) 17-epiestriol, an estrogen
metabolite, is more potent than estradiol in inhibiting vascular cell adhesion molecule 1
3
organization of the 17-OH and 18CH without changing the location
(
VCAM-1) mRNA expression. J Biol Chem 278:11746–11752.
of the 17-OH with respect to the plane of ring D (Fig. 6). The
glucuronidation results with estradiol and 13-epiestradiol suggest that,
with the exception of UGT2B17, the large change in the estradiol
molecule upon the configuration change at C13 does not affect the site
at which the different UGTs conjugate estradiol, but it does change the
reaction rate. The results with UGT2B17 were, however, surprising
because it also conjugated the 3-OH on ring A, an observation that has
never been reported before for UGT2B17 with any estrogen.
The results of this study shed new light on estrogen glucuronidation
by the human UGTs, particularly the structural components within the
estrogen substrate that, often differently, affect their glucuronidation
by individual UGT enzymes. These findings are expected to assist in
predicting the glucuronidation of drugs that resemble estrogens or
carry estrogen-like structural elements, such as the phase I metabolite
of exemestane (Sun et al., 2010). In addition, they will contribute to
a better understanding of the complex structure of the substrate-binding
site of the human UGTs and identification of the residues and other
structural properties that allow them to glucuronidate many different
substrates while retaining a considerable degree of specificity.
Ohno and Nakajin (2009) Determination of mRNA expression of human UDP-
S
S
glucuronosyltransferases and application for localization in various human tissues by real-
time reverse transcriptase-polymerase chain reaction. Drug Metab Dispos 37:32–40.
Radominska-Pandya A, Czernik PJ, Little JM, Battaglia E, and Mackenzie PI (1999) Structural
and functional studies of UDP-glucuronosyltransferases. Drug Metab Rev 31:817–899.
Ruggiero RJ and Likis FE (2002) Estrogen: physiology, pharmacology, and formulations for
replacement therapy. J Midwifery Womens Health 47:130–138.
Sneitz N, Court MH, Zhang X, Laajanen K, Yee KK, Dalton P, Ding X, and Finel M (2009) Human
UDP-glucuronosyltransferase UGT2A2: cDNA construction, expression, and functional charac-
terization in comparison with UGT2A1 and UGT2A3. Pharmacogenet Genomics 19:923–934.
Sneitz N, Krishnan K, Covey DF, and Finel M (2011) Glucuronidation of the steroid enantiomers
ent-17b-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucur-
onosyltransferases. J Steroid Biochem Mol Biol 127:282–288.
Starlard-Davenport A, Xiong Y, Bratton S, Gallus-Zawada A, Finel M, and Radominska-Pandya
A (2007) Phenylalanine(90) and phenylalanine(93) are crucial amino acids within the estrogen
binding site of the human UDP-glucuronosyltransferase 1A10. Steroids 72:85–94.
Sun D, Chen G, Dellinger RW, Sharma AK, and Lazarus P (2010) Characterization of 17-
dihydroexemestane glucuronidation: potential role of the UGT2B17 deletion in exemestane
pharmacogenetics. Pharmacogenet Genomics 20:575–585.
Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, ex-
pression, and disease. Annu Rev Pharmacol Toxicol 40:581–616.
Address correspondence to: Moshe Finel, CDR, Faculty of Pharmacy, P.O. Box
5
6 (Viikinkaari 5), FIN-00014 University of Helsinki, Finland. E-mail: moshe.finel@
helsinki.fi